Our laboratory (Unité d'Immunologie Virale) reached in 2005 its last (of 16) year of existence under its present configuration. Continuity of thematics and approaches, however, is ensured by the creation in 2006 of a new laboratory under the direction of F. Arenzana-Seisdedos . These thematics concern host-virus relationship, in virus diseases of great public health impact, such as AIDS and dengue infections, investigated at a critical stage of the virus cycle, namely the entry stage. For the last four years, focus was on interactions of virus envelopes and cell membranes. The global approach followed was to develop parallel and interactive investigations on the physiology and pathology of membrane receptors to chemokines and viruses. In 2005, the main virus investigated was dengue virus. The laboratory also concentrated efforts on the basic mechanisms of chemokine receptor endocytosis, and the relevance of this phenomenon to a human haematological and immunological disorder (WHIM syndrome), in an effort to follow "molecular medicine" approaches, the general thematics of the Pasteur department coordinated by the head of this laboratory (JLV)

The work described below was performed under the joint direction of F. Arenzana-Seisdedos and J-LVirelizier

We previously showed that the G-protein coupled receptor CCR5, the major HIV co-receptor, undergoes internalisation upon ligation of relevant CC chemokine agonists, a phenomenon shown to be the basis of the blockade by chemokines of HIV entry into cells (Amara et al, 1997).We now show that CCR5 displayed agonist-independent coupling to G-proteins. A CCR5 mutation on Arg 126 residue in the DRY motif abolished CCR5-mediated G-protein activation, but did not alter agonist-promoted phoshorylation and β-arrestin-dependent endocytosis. The mutant also displayed higher basal phosphorylation than wild-type CCR5. Induced expression of β-arrestin downregulated expression of the mutant receptor, suggesting spontaneous association of β-arrestins with the mutated receptor. However, migration of cells expressing the mutant receptor was not promoted by β-arrestin, contrary to what was seen in cells expressing wild type receptors. Altogether, these data indicate that different structural requirements for CCR5-mediated activation of G proteins are needed for β-arrestin-mediated chemotaxis, but not for receptor desensitisation and internalisation. Such dissociation is compatible with the concept that different biological responses of CCR5 may depend on different conformations sets of the receptor (Laganne et al, Mol. Pharmacol.,67, 1966, 2005)

- Characterisation of RDC1 as a novel , functional receptor for the chemokine SDF-1 (CXCL12)

Chemokines regulate many biological functions. The CXC chemokine SDF-1 (CXCL12) has a particularly large array of biological activities, although it was supposed to have only one receptor, CXCR4. We have shown that the orphan receptor RDC1, known to be used as entry co-receptor by some HIV and SIV strains, is able to bind to and be induced functionally by SDF-1. We demonstrated that RDC1 is expressed in T lymphocytes. Moreover, SDF-1-induced chemotaxis could be blocked by antibodies to RDC1. Interaction of RDC1 and SDF-1 was shown to be specific, saturable and of high affinity. In addition, we showed in CXCR4- negative, RDC1-positive cells that SDF-1 promotes internalisation of the receptor and chemotactic signals through RDC1. We have thus suggested that RDC1 is a novel SDF1 receptor, and proposed the name of CXCR7 for that receptor which is not any more orphan. (Balabanian, K., et al, J. Biol. Chem., 280,35766, 2005)

2- Pathological aspects of DC-SIGN and CXCR4 receptor function :

- DC-SIGN internalization signals are not involved in the use by dengue virus of this dendritic cell lectin as entry receptor

Responsible scientists : Lozach, P-Y, Amara, A., et al

The human cellular receptor able to initiate dengue virus (DV) primary infection upon skin inoculation by the rostrum of DV-infected mosquitos has long be an enigma. The first cell type to be infected is thought to be cells of the dendritic lineage, and indeed we previously showed that expression of the mannose lectin receptor DC-SIGN (Navarro-Sanchez et al, 2003) renders cells fully susceptible to DV infection . Using preparations of dendritic cells raised in vitro from circulating precursors, we found that the virus productively infects immature dendritic cells (DCs) but not Langherans cells (LCs) . Interactions between DC-SIGN and the virus envelope protein DV-E , the sole mannosylated glycoprotein present on DV particles, were shown to be essential for infections of DCs. Binding on DC-SIGN of mannosylated N-glycans present on the DV-E protein rapidly induces internalisation of DV-E. In contrast , we observed that endocytosis-defective DC-SIGN molecules allow efficient DV replication. This indicated that DC-SIGN endocytosis per se is dispensable for the internalization step of DV entry. Although these observations do not permit to conclude whether DC-SIGN acts as a cell surface attachment factor or as an authentic entry receptor mediating virus internalisation, they shed an interesting light on the first molecular events leading to receptor-mediated endocytosis of the DV flavivirus (Lozach, P-Y., et al, J. Biol. Chem., 280, 23698, 2005) .

Since SDF-1/CXCR4 interactions are known to control import , retention and exit of precursor haematopoietic cells from the bone-marrow, we were prompted to investigate the functioning of this chemokine/receptor couple in patients with the WHIM syndrome, showing a peripheral neutropenia contrasting with normal production of myeloid cells in the bone marrow. We studied patients from 3 different pedigrees. A genetic defect of CXCR4 ( mutation encoding a novel C-terminally truncated receptor) was found in two siblings, but no genetic abnormality of CXCR4 was found in two unrelated patients. These genetic differences were observed despite very similar clinical syndromes in patients from these different families. However, a common biological syndrome was characterised in all patients investigated , characterised by exaggerated G-protein-dependent CXCR4-mediated responses to SDF-1 in circulating lymphocytes and neutrophils. This phenomenon was found to be underlaid by a striking refractoriness of CXCR4 to be both desensitised and internalised in response to SDF-1. Enhanced response were not observed when using chemokines other than SDF-1, such as CCL4, CCL5 or CCL21. Thus, whether or not the CXCR4 gene itself was abnormal, CXCR4 functions, (but not that of other chemokine receptor tested) were found to be abnormal in all patients with the WHIM syndrome, thus defining a biological syndrome characteristic of that syndrome. We propose that permanently increased CXCR4 function(s), in particular excess retention of precursor cells in the bone marrow, account in vivo for the immuno-haematological disorder observed in patients with the WHIM syndrome. (Balabanian, K, et al, Blood, 105, 2449, 2005).

There is now a need to characterise the abnormal gene(s) necessarily involved in WHIM patients with no CXCR4 mutation, in order to explain the abnormal CXCR4 -dependent signalling . The WHIM syndrome also associates severe papilloma virus-induced condyloma. The complex functional relations between SDF-1/CXCR4 signals and tumorogenesis and angiogenesis is now being investigated (Orimo A. et al, Cell, 121, 335, 2005)

3- Signaling capacity of the HIV envelope glycoprotein

Responsible scientist : L. Chakrabarti

Within the frame of a multi-department project (GPH on HIV/AIDS), we are investigating whether the interaction of HIV-1 envelope glycoprotein gp120 with its coreceptors on CD4+ T lymphocytes can generate aberrant signal transduction cascades, which would contribute to the abnormal T cell activation characteristic of HIV-1 infection. We report that, while CXCR4-using gp120 signals like a full agonist, CCR5-using gp120 appears to induce a more restricted set of signals. We now aim to determine whether signaling through the coreceptor influences the routing of viral particles during early post-entry steps.

Legend to the figure:

Capture of fluorescent HIV particles by target cells

HIV-1 particles labeled with a GFP-Vpr fusion protein (green) are captured by cells expressing a DsRed-clathrin fusion protein (red). Some particles are captured by clathrin-coated pits (yellow dots) while other avoid the endocytic pathway (green dots).